Liquid-liquid extraction in an aqueous two-phase system (ATPS) can offer a powerful technique for isolation and purification of proteins. The separation of macromolecules and particles by means of liquid-liquid extraction is well known (Albertsson, 1986; Walter et al., 1985; Kula, 1990). Mainly polyethylene glycol (PEG)-salt, PEG-dextran and PEG-starch systems have been in use. More recently detergents and detergents with reversed solubility were discovered as suitable methods for separation of macromolecules, and especially for the separation of proteins.
An advantage of aqueous two-phase systems (ATPS) is that they are especially suited for large scale processing of microbial proteins not only from culture supernatants but also from crude extracts containing cells and cell debris (Kula, 1979; Kula, 1985). Characteristic features of biological fluids as well as suspensions are rather small particle sizes, low density differences between fluid and suspended solids, high viscosities of the extracts and high compressibility of the solids (Hustedt et al., 1985; Bender and Koglin, 1986). These attributes decrease the performance of conventional methods for solid-liquid separation like centrifugation and filtration at the beginning of a protein recovery process. Using an aqueous two-phase system removal of solids can be integrated into a liquid-liquid separation step, clarification is thus combined with an initial purification (Kula, 1979; Kula, 1985).
After the extraction process phase separation can be accomplished by settling under gravity as well as by centrifugation (Kula, 1985). ATPS can be applied in various scales from very small laboratory scale to large industrial scale thus suiting for various proteins, purposes and needs. With regard to industrial purposes commercially available centrifugal separators can be used to shorten separation time. Several authors have investigated the potential of centrifugal separators of various design for processing of large volumes of aqueous two phase systems (hula, 1979; Kula et al., 1981, Kula et al., 1982; Kula, 1985). In these studies the authors have used polymer/polymer or polymer/salt systems and the results of these investigations demonstrate the feasibility of continuous separation of aqueous two-phase systems in centrifugal separators.
Extraction systems based on nonionic surfactants have been described as an alternative to standard polymer/polymer or polymer/salt systems. Phase forming surfactants are e.g. polyoxyethylene type nonionic detergents. The basis of this type of aqueous two-phase system is the temperature-dependent reversible hydration of the polar ethylene oxide head groups. The temperature at which the phase separation occurs is referred to as the cloud-point (cloud-point extraction). This kind of aqueous two-phase system is especially suited for the extraction of amphiphilic biomolecules. The potential of this type of two-phase system for separating membrane bound proteins from cytosolic and peripheral membrane proteins was first demonstrated by Bordier (1981). Heusch and Kopp (1988) have been able to demonstrate that lamellar structures formed in the miscibility gaps of polyglycol ether/water systems are responsible for the selective extraction of hydrophobic substances.
Recently, the successful application of a surfactant-based aqueous two-phase system for the extraction of a membrane bound protein (cholesterol oxidase) from the unclarified culture medium of the gram-positive microorganism Nocardia rhodochrous on a bench scale has been reported (Minuth et al., 1995). By addition of only one chemical compound a product release through solubilization was possible in homogeneous phase and in a second step a clarification as well as an initial purification was achieved by an extraction process at elevated temperatures separating the detergent rich phase. A closed concept was further developed for the production of the membrane bound enzyme by surfactant-based extraction, organic solvent extraction and anion-exchange chromatography, which gave a product suitable for analytical applications (Minuth et al., 1996).
In aqueous two phase systems the desired target e.g. a protein should partition selectively into one phase (preferentially the lighter phase) while the other substances should partition into the other phase (preferentially the heavier phase). In PEG/salt and PEG/dextran and similar systems there are several driving forces for a substance like charges, hydrophobic, hydrophilic forces or the dependence on conformation or ligand interaction (Albertsson, 1986). The forces leading to separation in detergent based aqueous two phase systems are suggested to be primarily hydrophobic (Terstappen et al., 1993). Even if a lot of work has been carried out in the field of prediction in ATPS, none of the designed models provides a physical picture of the phase behaviour and prediction is hardly possible (Johansson et al., 1998).
In ATPS the partitioning coefficient is defined as the concentration (activity in case of an enzyme) of the target in the top phase divided by the concentration (enzyme:activity) of the target protein in the bottom phase. Partitioning coefficients in ATPS systems are usually in the range from less than 1 up to less than 100 (Terstappen et al., 1992; Terstappen et al., 1993).
  K  =            c              i        ,        T                    c              i        ,        B            
Yield: is defined as the amount of target in the top phase divided by the sum of the amount of target in top and bottom. This leads to the following equation
      Y    T    =      1          1      +              [                                            V              B                                      V              T                                ·                      1            K                          ]            
If the desired substance is directed to the heavier phase (as it can be the case using Triton) the yield is defined by
      Y    B    =      1          1      +              [                                            V              T                                      V              B                                ·          K                ]            
The volume ratio of the two coexisting phases are defined by the volumes of the lighter over the heavier phase, respectively.
  R  =            V      T              V      B      
An example of useful proteins facing problems in purification in a cost-effective way are the commonly used industrial enzymes used as biocatalysts, the glycosyl hydrolases, proteases and lipases produced by fungi and bacteria. These are used in e.g. laundry, textile, paper and pulp, food and feed industry. The fact that microbes produce many different enzymes during their growth and the fact that some of these may be undesired in certain applications leads to a need to enrich the active component(s). This enrichment can be performed by choosing appropriate growth conditions, by genetic engineering and/or by down-stream processing (e.g. purification of the active component(s)).
Purification of proteins are generally performed by chromatography. Usually gel-chromatographic methods are used based on ion-exchange, hydrophobic interaction, affinity chromatography and molecular sieving. Methods like electrophoresis and crystallisation can also be used. These methods are well known in the art and suitable for proteins of fairly high market value. In case of bulky enzyme production these methods, however, are too expensive in order to keep the final product on a compatible price level. Due to similar properties of these enzymes several purification steps are usually needed to separate the proteins from each other. This often causes low final yields and therefore a high loss of product.
Many extracellular hydrolases produced by the filamentous fungus Trichoderma are currently used in different industrial applications in large scale. These hydrolases are e.g. hemicellulases (such as xylanases and mannanases), cellulases (such as endoglucanases and cellobiohydrolases) and proteases. Purification of these is well known in the art (Bhikhabhai et al., 1984; Pere et al., 1995), but for large industrial applications the purification methods are too expensive. Alternative methods to enrich these hydrolases have been used, including deletion of undesired genes by genetic engineering (Suominen et al., 1992). However, even after extensive genetic engineering some minor undesired activities may still be present in the final product.
ATPS have been studied in purification of cellulases of T. reesei and the purification of an endoglucanase III showed some promising results, enriching the yield of the protein in the upper phase (U.S. Pat. No. 5,139,943). ATPS have also been studied in purification of lipases, endoxylanase and natamycin (EP 0 574 050 A1). No K and Y values are, however, mentioned.
As in other protein purification methods, similar properties of proteins produced by an organism impair also in ATPS, e.g. selective separation of one protein is not achieved optimally. To obtain selectivity in purification affinity chromatographic methods are used especially for analytic purposes and in purification of high-value products. These include immunoaffinity chromatography and various fusion protein strategies well described in the art such as fusing the protein of interest to an other protein (e.g. glutathione-S-transferase), protein domain (e.g. protein A-ZZ domain) or small peptide (e.g. His-tag), which selectively bind to the solid carrier and thus the recovery of the fusion partner is obtained as well. The fusion protein can be suitable for the particular purpose as such or cleavage of the product from the added fusion partner may be desired. There are well-known methods in the art on cleavage of fusion proteins from their partners by proteases, e.g. by factor X, thrombin or papain or by genetically introducing a protease cleavage site (e.g. Kex2 site) or autoprocessing domains (e.g. Intein, New England Biolabs) or by chemical cleavage (e.g. CNBr).
ATPS offer advantages mainly with respect to technology compared with the solid state based separation systems e.g. affinity column-based techniques. The scale-up of extractive enzymes is relatively simple utilising commercially available equipment and machinery common in the chemical industry. In addition, it can be used in a continuous process and it can be relatively cost extensive. It can be used as a single step for clarification, concentration and purification. ATPS can be used as a first capture-step, but for bulk products often no further purification is needed.
To aid selective separation in two-phase systems, recent publications have described the fusion of small peptide tags of 12 amino acids to the protein to be purified. The most successful of these soluble peptides are containing tryptophans. So far they have mainly been applied for very small molecules like the staphylococcal protein A derivative ZZT0 (Berggren et al., 1999; Hassinen et al., 1994; Köhler et al., 1991).
Use of ATPS has so far been limited to certain targets. Due to the advantages of ATPS in protein separation, purification and localisation, highly selective and powerful methods should be developed. This is especially important for large scale processes where ATPS in general is very inexpensive as a first capture step or as the only step for purification, clarification and concentration. The system should be universal so that the technique would be strong enough to mediate separation of in principle any component to the desired phase irrespective of its size or biochemical properties.